Abstract

Ornithopus sativus Brot. (French serradella, pink seradella) is an annual legume in the Leguminosae sub-family Papilionoideae that has value in dryland agriculture as a forage or green manure. It is cultivated in Mediterranean and temperate climates where it is favored for the production of high quality fodder, robust nitrogen fixing symbiosis, and adaptation to acidic and relatively infertile soils. In common with grain legumes, cultivated forms of O. sativus are soft-seeded, while wild relatives of the genus are generally hard-seeded. The dynamics of these opposite seed characteristics are explored in O. sativus along with the environmental control over the initiation of flowering.

In two cultivated populations of O. sativus, rare hard-seeds which did not imbibe moisture were isolated. Plants (S1) grown in an open field from these residual hard-seeds produced either all soft-seeds (0% hard-seed) or a proportion of hard-seeds (4 – 100%). The offspring (S2) from a subset of these plants showed a similar distribution and correlation between parent and offspring production of hard-seeds. However, the proportion of hardseed produced and the parent-offspring relationship was different for the two source populations.

The hard-seeds selected from accession 97ZAF5sat produced S1 plants expressing the extremes in hard-seed production and S2 plants segregating to the opposite expression of their maternal parent. All of these selections commenced flowering relatively early with little variation between individuals and no apparent association of timing of flowering with hard-seed production.

Plants grown from the hard-seeds selected from cv. Emena also produced either all soft-seeds or some proportion of hard-seeds. However, among the hard-seed producing individuals there was a broad range in the proportion of hard-seeds produced and there was less segregation away from the maternal expression in a subsequent generation of offspring (compared to the 97ZAF5sat selections). There was also a broad range in the timing of flowering in the selections from cv. Emena and a positive correlation between the timing of flowering with the proportion of hard-seed produced by both the S1 and S2 generations.

The variation observed in the O. sativus populations studied for hard-seed production and timing of flowering (both between individual plants and the segregation away from maternal plant expression) was the result of cross pollination. Between-plant cross pollination in O. sativus, in an open field with honey-bee activity, was measured at 25.3% (± 0.8). This was assessed by examining the pollen flow from pink-flowered plants to white-flowered maternal plants. This result demonstrated that although O. sativus is a selfcompatible species, it should be considered to have a mixed breeding system with allogamic cross pollination possible in the presence of pollinating insects.

The inheritance of seed coat permeability was examined by hybridization between plants that were homozygous for their respective seed character. A hard-seed parent plant was selected from each of the 97ZAF5sat (S1 plant A1.1) and cv. Emena (S1 plant B1.2) populations based on consistent hardseed production in S2 offspring. Hybrids were generated between these plants and also with two soft-seeded parents. One of the soft-seeded parents was selected from cv. Cadiz (C) and other was a very early flowering selection from this cultivar (D1). Sequential planting of these parent plants in a controlled temperature glasshouse showed variation in the timing of flowering to be control by a basic accumulation of days (at constant temperature) that is modified by photoperiod. Differences between the four genotypes in the timing of flowering were generated by inhibition of flowering under short photoperiods. In the early flowering selection, D1, the influence of photoperiod was reduced.

All F1 seed from hand pollination was either soft-seeded or hard-seeded depending on the phenotype of the maternal parent. This confirmed that the permeability of a seed coat (a maternal tissue), is determined by the genotype of the maternal plant, regardless of the genotype of the seed embryo. F1 hybrid plants with A1.1 maternal or paternal parentage produced very high proportions of hard-seeds (greater than 80%) regardless of the expression of the other parent (hard or soft-seeded). However, the hybrid between a soft-seeded D1 maternal plant and a paternal hard-seeded B1.2 produced soft-seeds.

Selfed F2 hybrids between hard-seeded A1.1 and soft-seeded parents segregated to 26.6% (± 1.3%) of plants producing all soft-seeds and the remainder producing high levels of hard-seed (>80% hard-seed). All selfed F2 hybrids between hard-seeded A1.1 and hard-seeded B1.2 produced hardseed, however the proportion of hard-seed produced by these hybrids ranged between the two parent expressions, with the majority producing the high percentages observed in A1.1. The F2 hybrids between B1.2 and D1 segregated to 77% of plants producing soft-seed and 23% of individuals producing some proportion of hard-seed (12% to 100% hard-seed).

The segregation evident in F2 hybrids for hard-seed production between A1.1 and the soft-seeded genotypes predicts its type of hard-seededness is conferred by a single dominant allele. Hybrids between the other hardseeded parent, B1.2, and the soft-seeded D1 suggest the inheritance of a single recessive locus being associated with hard-seed production. Further, the F2 hybrids between the two hard-seeded genotypes showed a dominance of A1.1 type hard-seededness. However, there was no obvious truncation point across the variation among these individual F2 hybrids to facilitate estimation of a segregation ratio.

The simplest model of inheritance of the hard or soft-seed expression is a single, qualitative locus with three possible alleles. In order of dominance; SCP1 > SCP2 > SCP3. SCP1 is associated with the hard-seed character selected from accession 97ZAF5sat which is expressed as high proportions of hard-seeds (impermeable seed coats) with little variation among individuals; SCP2 is associated with soft-seed production and all seed is permeable to moisture; and SCP3 is associated with the hard-seed character selected from cv. Emena, expressed as variable proportions of hard-seed between individuals.

Seeds produced by the soft-seeded genotype D1 freely and rapidly exchanged moisture with the environment, and this exchange was not influenced by seed moisture content. Staining revealed areas permeable to moisture at sites randomly distributed over the whole seed coat, but not at the lens or hilum regions.

Hardseededness of the A1.1 genotype was stable at a moisture content of 6.6% (seed DM) and the number of hard-seeds did not change with further drying. Re-adsorption of moisture at 76% relative humidity was minimal in these seeds at this, or drier seed moisture contents. Staining revealed that when hard-seed breakdown is induced, these seeds first absorb moisture through the lens, and then through gradual hydration and expansion of the seed coat radiating away from the lens.

The number of hard-seeds in seed lots produced by Genotype B1.2 progressively increased as they were dried to approximately 4% moisture content. Above 4%, the seeds displayed a protracted imbibition and germination pattern. Staining showed that moisture was absorbed by these seeds at sites randomly distributed over the whole seed coat, but not at the lens or hilum regions. However, below 4% moisture content, when hard-seed breakdown was induced, these seeds developed a breach at the lens region and moisture uptake and germination was rapid.

Seasonal changes in seed germination were measured by exposing seed (in lomenta) in mesh pockets either on the soil surface or covered with 1cm of soil. Three cultivars of O. sativus, Cadiz (soft-seeded), Erica (hard-seeded) and Margurita (hard-seeded) were compared along with a hard-seeded cultivar of O. compressus (Santorini) (yellow serradella). The seed was produced at two sites with different soil types. The seed germination behaviour of cv.s Erica and Margurita are best described by the dominant SCP1 type hard-seed character, and therefore the recessive SCP3 type hardseed was not represented in this experiment. Seed produced by the softseeded cultivar deteriorated on the soil surface, but this was reduced when seed was slightly covered with soil. Breakdown of hard-seeded cultivars determined that germination was distributed across at least three growing seasons after the year of production.

The single locus, three allele concept of inheritance was applied in a stepwise model to predict generational/seasonal population changes in hard and soft-seed production. Biological and environmental factors included in the model include initial (fresh) germination, cross-pollination, changes in seed germination in situ (seasonal hard-seed breakdown, seedling viability), and frequency of seasonal seed production. An important consideration in the model is the generational lag that occurs due to the control of the maternal phenotype on seed germination, particularly when allogamic cross pollination occurs.

In the absence of conditions or treatments to break hard-seed dormancy, the model predicts O. sativus populations, cultivated in the traditional way of repeated harvesting for re-sowing, will rapidly become dominated by softseeded genotypes. The rate of loss of a dominant form of hard-seededness will be greater than for a recessive form. In both cases, the rate of change is reduced by cross-pollination.

Selective pressure favors hard-seed production when O. sativus is left to self-regenerate in situ. In a permanent pasture, the rate of change in a mixed hard and soft-seed producing population will, in the short term, be determined by the balance between seed deterioration of soft-seeded phenotypes and the rate of hard-seed breakdown (until soil seed reserves reach an equilibrium). As the hard-seed reserve in the soil approaches equilibrium, the selective pressure is driven wholly by rate of deterioration in soft-seeds.

The greatest rate of change in a population structure occurs with reproductive failure in a single or series of reproductive cycles. In these scenarios, the rate of change is more rapid with the recessive S3 type hardseed than S1, because the dominant effect protects the allele for soft-seed. Cross-pollination reduces the rate of change in both cases of hard-seed. However, because of its absolute expression, all soft-seed alleles will be removed from a population with a single season of failed reproduction in the absence of heterozygosis and cross-pollination.

This research showed that there are two genetically-controlled forms of hardseed in O. sativus, each with a different expression of inheritance, and interaction with seed moisture. This has implications for the management of hard-seedness in O. sativus and in other legume species where moderate or variable proportions of hard-seeds occur. To maintain a proportion of hardseeded genotypes in a cultivated population, that is sown and harvested each year, the harvested seed must be treated to remove the hard-seed dormancy before re-sowing.

The opportunities to expand the use of O. sativus in agricultural systems are discussed along with appropriate protocols that could be used in O. sativus breeding programs to achieve the full potential of the species.